During normal aging, cellular enzymes accumulate as catalytically inactive or less active forms. The mechanisms regulating these changes, as yet unknown, may involve mixed- function oxidation of critical amino acid residues. In an effort to define the mechanisms responsible for these alterations, we have developed an in vivo model system to compare the protein changes observed during aging with changes mediated by exposure of the animals to an acute oxidative stress. We have examined the levels of oxidized proteins, enzyme inactivation, and protease activity in hepatocytes isolated from rats exposed to 100% oxygen or from rats of various ages. The levels of oxidized protein increase with animal age and also with exposure to 100% oxygen up to 48 hours followed by a sharp decrease between 48 and 54 hours. A decrease in the specific activity of glutamine synthetase and glucose-6-phosphate dehydrogenase was observed under each experimental condition and preceded the loss of immunological cross reactivity indicating the accumulation of inactive proteins. Hepatic protease activity at pH 2.5, 5.5, 7.4, and 8.8 was assayed in each sample. The acid proteases showed little variation under both conditions; however, the alkaline protease activity decreases with age and remains constant during the first 48 hours of oxygen exposure with a significant increase in activity observed between 48 and 54 hours. Alkaline protease activity correlated inversely with accumulation of oxidized protein. These results invite speculation that the accumulation of oxidized protein and the inactivation of enzymes which occur when animals are exposed to a high concentration of oxygen may reflect an acceleration of the same metabolic changes that lead to the accumulation of oxidized proteins and enzyme inactivation during aging and accelerated aging diseases.